The HIV-1 surface glycoprotein Env (gp120/gp41) mediates viral entry through a series of coordinated structural changes initiated when CD4 interacts with gp120. Ultimately, the gp41 ectodomain folds into a compact trimer-of-hairpins that brings the viral and cellular membranes into the close proximity required for efficient membrane fusion. These conformational transitions have been delineated, in part, through the use of C- peptide and 5-Helix inhibitors that target the HR1 and HR2 regions of gp41 and block trimer-of-hairpins formation. Our previous work has described the physical basis behind C-peptide and 5-Helix inhibition and quantitatively modeled the relationship between inhibitory potency and fusion kinetics. The experiments outlined in this application explore the structure and dynamics of Env in its native and intermediate conformations. The research will address three fundamental questions regarding the mechanism of viral membrane fusion: How does CD4 binding to gp120 trigger formation of the prehairpin intermediate state? What is the structure of Env in this intermediate conformation? What aspects of gp41 folding from the prehairpin state drive the fusion of viral and cellular membranes? Owing to the unstable nature of Env in its native and intermediate states, these questions are often difficult to address using standard experimental techniques, such as scanning mutagenesis, that require stable proteins in long lived conformations. Our basic approach will be to generate new inhibitors that target the HR1 and HR2 regions, and dissect the mechanism of resistance to them. This strategy takes advantage of the power of natural selection to generate well-behaved mutant Env variants with interesting structural or dynamic properties. The specific aims are: 1) to explore the structure and exposure of the HR1 region using novel inhibitors engineered to overcome standard C-peptide resistance profiles; 2) to probe the structure and exposure of the HR2 region using multivalent inhibitors, and to dissect the mechanisms of resistance to these agents; and 3) to explore the energetic requirements of Env- mediated membrane fusion, and to examine the structure of mutant Env variants trapped in intermediate conformations. Our findings will provide new insights into the dynamic properties of the gp120/gp41 complex critical to the viral entry mechanism and will aide the development of new viral entry inhibitors and HIV-1 vaccines.